Chemical Kindling Model Research Articles

P/Q type (Cav2.1) Calcium Channel Blocker ω-Agatoxin IVA Alters Cleaved Caspase-3 and BDNF Expressions in the Rat Brain and Suppresses Seizure Activity.

High-voltage-gated calcium channels have pivot role in the cellular and molecular mechanisms of various neurological disorders, including epilepsy. Similar to other calcium channels, P/Q-type calcium channels (Cav2.1) are also responsible for vesicle release at synaptic terminals. Up to date, there are very limited reports showing the mechanisms of Cav2.1 in epileptogenesis. In the present study, we investigated the anticonvulsive and neuroprotective effects of ω-agatoxin IVA, a specific Cav2.1 blocker, in a chemical kindling model of epileptogenesis. Righting reflex and inclined plane tests were used to assess motor coordination. Electroencephalography was recorded for electrophysiological monitoring of seizure activity in freely moving rats. Immunohistochemical analyses were performed for brain-derived neurotrophic factor (BDNF) and cleaved caspase-3 expressions in the prefrontal cortex, striatum, hippocampus, and thalamic nucleus. ω-Agatoxin IVA injected into the right lateral ventricle significantly prolonged the onset of seizures in a dose-dependent manner. In addition, repeated intraperitoneal administrations of ω-agatoxin IVA significantly suppressed the development of kindling and epileptic discharges without altering motor coordination. In addition, ω-agatoxin IVA significantly increased BDNF expressions, and decreased cleaved caspase-3 expressions in the brain when compared to PTZ + saline group. Our current study emphasizes the significance of the inhibition of P/Q type calcium channels by ω-agatoxin IVA, which suppresses the development of epileptogenesis and provides a new potential pathway for epilepsy treatment.

Neuroprotective Effect of Celastrus Paniculatus Seed Extract on Epilepsy and Epilepsy-associated Cognitive Deficits.

Cognitive deficit is one of the common comorbidity accompanying epilepsy. The present study evaluated the effect of Celastrus paniculatus seed extract on seizure severity and cognitive deficit following the pentylenetetrazole (PTZ)-induced chemical kindling model. PTZ kindling model was developed by daily administration of the sub-convulsive dose of PTZ 30 mg/kg for four weeks. After four weeks of induction, the following treatment, namely sodium valproic acid (SVA) 200 mg/kg, C. paniculatus 500 mg\kg, pergolide 2 mg/kg, C. paniculatus (250 mg\kg)+ Pergolide (1 mg/kg), and C. paniculatus (250 mg\kg)+ SVA (100 mg/kg) were administered 30 minutes prior to PTZ (30 mg/kg) injection for a period of next 14 days. Neurobehavioral parameters, including superoxide dismutase (SOD), Catalase (CAT), glutathione (GSH), and dopamine levels were assessed and the Morris water maze test (MWM) and Grip strength test (GPS) were performed. Hematoxylin & Eosin (H&E) staining of hippocampal cornu ammonis (CA1), CA2, CA3, dentate gyrus (DG), and frontal cortex was performed. C. paniculatus (500 mg/kg) alone and in combination (C. paniculatus (250 mg\ kg)+ pergolide (1 mg/kg) and C. paniculatus (250 mg\kg)+ SVA (100 mg/kg)) significantly (P<0.05) reduced the seizure score, mean latency time, and distance traveled in the MWM. However, no significant effect was seen in GPS. Biochemical analysis showed elevated antioxidant markers, namely GSH, CAT, and SOD, and also elevated dopamine levels. C. paniculatus and its combination also significantly (P<0.05) protected against neuronal loss in the hippocampus and frontal cortex evidenced by H&E staining. C. paniculatus alone and in combination with other agents may have the potential to treat epilepsy and associated cognitive deficits.

Ameliorating Effects of Dorema ammoniacum on PTZ-Induced Seizures and Epileptiform Brain Activity in Rats.

The objective of the current study was to investigate the anti-epileptogenic and anticonvulsant effects of Dorema ammoniacum gum, which is used in Iranian traditional medicine for the treatment of seizures. Animals received pentylenetetrazol (IP, 30 mg/kg/48 h) for inducing seizures. Five different seizure stages were evaluated for 20 min and parameters including maximum seizure stage, the latency to the onset of stage 4, stage 4 duration, and seizure duration were measured. D. ammoniacum (50 and 100 mg/kg) or its vehicle was administered 30 min before or after pentylenetetrazol injection in different groups. In addition, the effective dose of D. ammoniacum (100 mg/kg) on different seizure stages was compared with the common antiseizure drug phenobarbital. In another set of experiments, we investigated the effective dose of D. ammoniacum on fully kindled animals in which an interictal electroencephalogram was recorded by superficial electrodes placed on the skull. The results showed that D. ammoniacum administration, before and after pentylenetetrazol injections, significantly decreased seizure stage, seizure duration, stage 4 duration, and 1/stage 4 latency. The anti-epileptogenic effect of D. ammoniacum was about 50 to 60% of phenobarbital. In addition, D. ammoniacum significantly decreased seizure stage, seizure duration, stage 4 duration, and 1/stage 4 latency when administered to fully kindled animals but had no effect on the power of EEG sub-bands. These results indicate that D. ammoniacum has anti-epileptogenic and anticonvulsant effects in a chemical kindling model of seizures.

Rational design of ultra-small photoluminescent copper nano-dots loaded PLGA micro-vessels for targeted co-delivery of natural piperine molecules for the treatment for epilepsy

In recent days, reported researches demonstrated that encapsulation of natural hydrophobic drug molecules (Piperine) into the biodegradable polymer system with nanoformulations opens a novel prospect in bio-nanomedicine field. Generally, the nanostructured materials embedded with the drug molecules could render enhanced efficiency in therapies. Piperine is a chief alkaloid compound of natural black pepper exhibits excellent anti-convulsant efficiency in the anti-epileptic treatment. Nonetheless, the poor water solubility of the piperine molecules has some difficulties in drug delivery and clinical applications. Herein we report the synthesis of Copper oxide quantum dots coated Hyaluronic acid (HA)/ Poly(lactic-co-glycolic acid) (PLGA) with for the effective delivery of piperine in the targeted drug delivery for epilepsy treatment. The physicochemical characterization was performed using the as prepared material. The crystal structure, surface morphology and the elemental composition were investigated from XRD, SEM, TEM and EDX analyses respectively. The surface morphology clearly stated the loading of CuO QDs loaded HA/PLGA microspheres. The capping of the polymer matrix was also studied using FTIR analysis. A Photoluminescence spectrum is also recorded. This study was illustrating that Piperine loaded CuQDs@HA/PLGA nanostructures exhibit improved neuroprotection and encourage the activation of astrocytes in chemical kindling model of epilepsy. This proposed material could be a novel and effective therapeutic platform for the targeted drug delivery systems.

Ursin reduce neuronal loss and astrocytes activation in chemical kindling model of epilepsy

Ursin reduce neuronal loss and astrocytes activation in chemical kindling model of epilepsy

Decreased epileptogenesis in mice lacking the System xc - transporter occurs in association with a reduction in AMPA receptor subunit GluA1.

SummaryObjectiveAlthough the cystine/glutamate antiporter System xc − (Sxc −) plays a permissive role in glioma‐associated seizures, its contribution to other acquired epilepsies has not been determined. As such, the present study investigates whether and how Sxc − contributes to the pentylenetetrazole (PTZ) chemical kindling model of epileptogenesis.MethodsMale Sxc − null (sut/sut) mice and their wild‐type littermates were administered PTZ (i.p.) daily for up to 21 days (kindling paradigm). Seizure severity was scored on a 5‐point behavioral scale. Mossy fiber sprouting, cellular degeneration, and Sxc − light chain (xCT) messenger RNA (mRNA) were explored using Timm staining, thionin staining, and real‐time quantitative polymerase chain reaction (qPCR), respectively. Levels of reduced and oxidized glutathione and cysteine were determined via high‐performance liquid chromatography (HPLC). Plasma membrane protein levels of glutamate and γ‐aminobutyric acid (GABA) receptor subunits as well as the K+/Cl− co‐transporter KCC2 were quantified via western blot analysis.ResultsRepeated administration of PTZ produced chemical kindling in only 50% of Sxc − null mice as compared to 82% of wild‐type littermate control mice. Kindling did not result in any changes in xCT mRNA levels assessed in wild‐type mice. No cellular degeneration or mossy fiber sprouting was discernible in either genotype. Except for a small, but significant, decrease in oxidized cysteine in the hippocampus, no other change in measured redox couples was determined in Sxc − null mice. Cortical levels of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor subunit GluA1 were decreased in Sxc − null mice as compared to wild‐type littermates, whereas all other proteins tested showed no difference between genotypes.SignificanceThis study provides the first evidence that Sxc − signaling contributes to epileptogenesis in the PTZ kindling model of acquired epilepsy. Further data indicate that a reduction in AMPA receptor signaling could underlie the resistance to PTZ kindling uncovered in Sxc − null mice.

Effect of dimethyl fumarate on neuroinflammation and apoptosis in pentylenetetrazol kindling model in rats

ObjectiveRole of apoptosis and neuroinflammation have been well established in the pathogenesis of epilepsy. It has been reported that the activation of nuclear factor-erythroid 2-related factor-2 (Nrf2) contributes to the attenuation of inflammation by inhibiting nuclear factor-kB (NF-kB) pathway. Therefore, the present study was designed to evaluate anti-inflammatory and anti-apoptotic role of dimethyl fumarate (DMF), an activator of Nrf2, in chemical kindling model in rats. MethodsChemical kindling model was established in Wistar rats by intraperitoneal (i.p.) administration of pentylenetetrazole (PTZ). Animals were treated with DMF (60 mg/kg) to activate the Nrf2 antioxidant response element (ARE) pathway. The animals were assessed for seizure score, neuronal damage and inflammatory cytokines levels (IL-1β, IL-6 and TNF-α) in hippocampus. The mRNA levels of various genes (Nrf2, HO-1, NQO1, Bcl2, Bax, Caspase 3, NF-kB, IL-6, IL-1β and TNF-α) were quantified by real-time PCR. The expression of anti-oxidative (Nrf2), apoptotic (Bax, Bcl2) and inflammatory (NF-kB) proteins were analysed by western blot. Immunohistochemistry (Bax) and electron microscopy were done to assess apoptosis. ResultsThe results showed reduction in the seizure score, percentage of kindled rats and neurological damage score in DMF treated rats. Pro-inflammatory cytokines concentrations were also decreased by DMF treatment. DMF downregulated the expression of inflammatory (NF-kB) and apoptotic (Bax, Caspase-3) genes and protein. DMF treatment increased the gene expression of Nrf2, HO-1, NQO1, Bcl-2 and protein expression of Nrf2 and Bcl2. ConclusionDMF demonstrated anti-apoptotic, anti-inflammatory and anti-oxidative effect in hippocampus, which might be regulated by increased level of antioxidant response elements.

Neuroprotective effect of Nrf2 activator dimethyl fumarate, on the hippocampal neurons in chemical kindling model in rat

BackgroundNuclear factor erythroid-2 related factor 2 (Nrf2) is a transcription factor, which activates the anti-oxidative stress response pathway. In epilepsy, oxidative stress is one of the key factors in the progression of the disease. So, the neuroprotective role of dimethyl fumarate (DMF), a Nrf2 pathway activator was explored in pentylenetetrazole (PTZ) induced kindling model of epilepsy in rats. MethodsMale Wistar rats were subjected to different doses of DMF (15, 30, 60 mg/kg) to evaluate the effect on the PTZ induced kindling in rats. Seizure scoring, the percentage of animals kindled, histopathological analysis of hippocampus and biochemical estimation were done to study the effect of DMF on PTZ induced oxidative stress in rats. Key findingsThe number of kindled animals in the DMF 60 mg/kg treatment group (25%) was less in comparison to corn oil + PTZ control group (62.5%). The histopathological analysis of the hippocampus revealed a significant (p = 0.003) decrease in neuronal damage in DMF 60 mg/kg group as compared to corn oil + PTZ control group. The DMF 60 mg/kg treatment improved the level of antioxidants: glutathione peroxidase (GPx), superoxide dismutase (SOD), reduced glutathione (GSH) and reduced the lipid peroxidation as compared to corn oil + PTZ group. ConclusionsDMF has demonstrated the neuroprotective effect in PTZ induced kindling model of epilepsy. This can prove advantageous in the development of new treatment strategies for epilepsy.

Potentiation of pentylenetetrazole-induced neuronal damage by dimethyl sulfoxide in chemical kindling model in rats.

OBJECTIVES:Dimethyl sulfoxide (DMSO) is commonly used as a vehicle for many hydrophobic drugs. This study aimed at evaluating the effect of low dose of DMSO (0.1%) on Pentylenetetrazole(PTZ) induced neuronal damage in rats.MATERIALS AND METHODS:Young male Wistar rats (n = 32) were divided into four groups as follows: saline control group, DMSO control group, PTZ group (35 mg/kg), and combination group (DMSO + PTZ). Animals were observed for seizure score, latency to develop kindling, percentage of animals kindled, and histopathological score of hippocampus.RESULTS:There was a significant increase in the seizure scores and histopathological scores in the combination group as compared to PTZ-treated group. The latency to develop kindling was, however, decreased in the combination group (4th week) as compared to PTZ (6th week) group.CONCLUSIONS:The present study has concluded that 0.1% DMSO in PTZ-induced rat model of epileptogenesis needs further optimization and should be used cautiously

Manual acupuncture improves parameters associated with oxidative stress and inflammation in PTZ-induced kindling in mice.

The use of acupuncture in the treatment of central nervous system (CNS) disorders is an age-old practice. Although only a few studies have proved its efficacy, evidence has indicated the use of acupuncture to treat different types of seizures. Therefore, the present study aimed to evaluate the effect of manual acupuncture (MAC) using the chemical kindling model. The role of MAC in oxidative stress and inflammation after pentylenetetrazole (PTZ)-induced kindling was investigated by measuring reactive oxygen species (ROS) production, superoxide dismutase (SOD), and catalase (CAT) activities, nitrite content, and deoxyribonucleic acid (DNA) damage in cerebral cortex. Mice received PTZ (60mg/kgs.c.) once every three days for 16days, totaling six treatments. MAC was applied at acupoint GV20 daily during the entire experimental protocol. Diazepam (DZP) (2mg/kg) was used as positive control. Also, we evaluated the MAC effect associated with DZP (MAC/DZP) at a low dose (0.15mg/kg). The results demonstrated that MAC or MAC/DZP were not able to reduce significantly seizure occurrence or to increase the latency to the first seizure during treatment. MAC/DZP promoted a difference in the first latency to seizure only on the third day. PTZ-induced kindling caused significant neuronal injury, oxidative stress, increased DNA damage, nitric oxide production, and expression of the pro-inflammatory Tumor Necrosis Factor-α (TNF-α). These effects were reversed by treatment with MAC or MAC/DZP. These results indicated that the stimulation of acupoint GV20 by MAC showed no potential antiepileptogenic effect in the model used, although it greatly promoted neuronal protection, which may result from antioxidant and anti-inflammatory effects observed here.

Piperine-loaded chitosan-STPP nanoparticles reduce neuronal loss and astrocytes activation in chemical kindling model of epilepsy

Recent evidence suggests that encapsulation of hydrophobic drugs in biodegradable polymers opens a new horizon in nanomedicine filed. Piperine, a main alkaloid form of black pepper possesses potent anticonvulsant activity. However, the low water solubility of piperine has limited its clinical application. In this study, piperine was loaded on chitosan-sodium tripolyphosphate nanoparticles (CS-STPP NPs) and the effect of piperine NPs on seizures behavior and astrocytes activation was assessed in pentylentetrazol (PTZ)-induced kindling model. Animals have received the daily injection of free piperine or piperine NPs at doses of 5 or 10mg/kg, 10days before PTZ injections and their intraperitoneally (i.p) administration continued until the last PTZ injection. The neuroprotective effects of piperine NPs were evaluated using nissl staining and immunostaining against NeuN. Astrocytes activation was examined by GFAP immunostaining. Behavioral data showed that piperine NPs have inhibited the development of seizure parameters compared to the free piperine groups. In addition, the levels of cell loss and astrocytes activation were reduced in piperine NPs groups. In conclusion, these data suggest that piperine NPs enhance the neuroprotection and ameliorate the astrocytes activation in chemical kindling model of epilepsy. This may provide an effective therapeutic strategy for the treatment of epilepsy disorder.

To evaluate the anti-kindling effect of allopregnanolone alone and its interaction with sodium valporate in pentylenetetrazole induced kindling model

Background and purposeStudies in the animal models of epilepsy have suggested the anti-seizure effects of neuroactive steroids and its derivatives in kainic acid and pilocarpine induced limbic seizures and status epilepticus in mice, but no such studies have been reported in the published literature on the role of allopregnanolone in chemical kindling model and its interaction with sodium valproate. The purpose of this study was to evaluate the interaction between sodium valproate and allopregnanolone in pentylenetetrazole induced kindling model in rats. MethodsIn a PTZ kindled Wistar rat model, sodium valproate and allopregnanolone were administered 30min before the PTZ injection. The PTZ injection was given on alternate day till the animal became fully kindled or till 10 weeks. The parameters measured were latency to develop kindling and incidence of kindling, histopathological study of hippocampus, hippocampal anti-oxidant parameters and hippocampal DNA fragmentation studies. ResultsIn this study, the combination of low dose of allopregnanolone with low dose of sodium valproate showed a similar beneficial effect to that of a higher dose of sodium valproate in significantly reducing the number of kindled animals (0/8) as compare to PTZ control group (5/8) as well as the seizure scores and the histopathological scores. The combination significantly reduces oxidative stress by significantly decreasing the MDA levels, and increasing the SOD levels and GSH levels in the hippocampus of rats as compared to PTZ control group. So all these data suggest the antiepileptic effect of the combination and confers the synergistic interaction between the allopregnanolone and sodium valproate. ConclusionsIt can be concluded that by choosing this combination the dose of sodium valproate can be reduced and thereby reduces the incidence of adverse effects caused by sodium valproate and hence proves to be a useful combination clinically. This study has lead the basis to further investigate the various combinations of neurosteroids and valproate in the process of epileptogenesis with better side effect profile.

Increased Go activity in C57Bl/6J mice enhances sensitivity to a model of epilepsy

Many patients suffering from temporal lope epilepsy (TLE) are refractory. This work focused on understanding two potential new therapeutic targets, the hetero‐trimeric G‐proteins Go and Gi2. We hypothesized that increasing activity of these proteins would be protective from seizures as inhibition of these proteins enhances seizure susceptibility. Mouse lines were created with increased activity of Go (Gαo RGSi) or Gi2 (Gαi2 RGSi) through the introduction of a point mutation in the α subunit. Both mouse lines were exposed to a chemical kindling model with relevance to TLE. C57Bl/6J Gαo+/RGSi mice have a faster onset to seizure while Gαi2+/RGSi mice have no change compared to controls. 129SvImJ Gαo+/RGSi mice however, kindle no faster than controls. Furthermore, a 129 modifier region on Chromosome 17 reduces the rate at which B6 Gαo+/RGSi mice kindle. Here we have demonstrated an in vivo role for Go and a novel modifier region in regulating susceptibility to a kindling model of epilepsy however we have yet to identify if these effects are through developmental or acute signaling changes. Supported by NIH grant RO1‐GM39561 (RRN), 5T32GM008322–19 (JMK) and PhRMA Foundation Fellowship to JMK

Increased Gαo activity in C57Bl/6J mice enhances sensitivity to a model of epilepsy

Despite a large number of anti‐epileptic drugs on the market, many patients suffering from partial complex epilepsy with secondary generalizations (PCE) are refractory to current medications. Thus identification of new therapeutic targets is required. This work focused on two targets, Gαo and Gαi2. We hypothesized that increasing activity of these proteins would be protective from seizures as inhibition of these and related proteins by pertussis toxin enhances seizure susceptibility in a pilocarpine model. Mouse lines were created with increased activity of Gαo (Gαo RGSi) or Gαi2 (Gαi2 RGSi). Both mouse lines were exposed to a chemical kindling model with relevance to PCE using repeated injections of pentylenetetrazol (35mg/kg, i.p.) three times per week. Gαo +/RGSi mice have a faster onset to seizure on the C57Bl/6J background while Gαi2 +/RGSi mice have no change compared to littermate controls. The enhanced kindling in Gαo +/RGSi mutant mice is background dependent, whereby Gαo +/RGSi mice on the 129SvImJ background kindle no faster than their littermate controls. Furthermore, a 129SvImJ modifier locus that we previously identified on Chromosome 17 that reduces early lethality of Gαo +/RGSi mice also reduces the rate at which Gαo +/RGSi mice kindle on the C57Bl/6J background. (Supported by NIH R01 GM036592‐23 and a fellowship from the PhRMA Foundation)

CCK-8 induces NGF and BDNF synthesis and modulates TrkA and TrkB expression in the rat hippocampus and septum: Effects on kindling development

In our previous studies, we demonstrated that intraperitoneal (i.p.) injections with the neurotransmitter/neuromodulatory peptide Cholecystokinin-8 (CCK-8) stimulate the synthesis of the neurotrophin nerve growth factor (NGF) resulting in the structural and functional recovery of neuronal damage. This neurotrophin-mediated neuroprotective action of CCK-8 has opened a new perspective for a better understanding of the CCK neurobiological and pharmacological properties. To explore the possible beneficial effects of the CCK-induced increase of neurotrophin availability in brain, we compared the effects of i.p. CCK-8 in healthy rats and in a chemical kindling model using a subconvulsive dose of pentylenetetrazol (PTZ). Behavioural changes were monitored during treatment and classified according to a six-point scale. After 3 weeks of treatment (12 trials), the PTZ group of rats manifested generalized clonic–tonic seizures (Class 5 behaviour). For this reason, this time point was chosen to compare the effects of CCK-8 treatment on the expression of NGF, the brain derived neurotrophin factor (BDNF) and their receptors in the septum and hippocampus. We found that repeated i.p. injections with CCK-8 in adult rats result in: (1) an increase of NGF and BDNF protein and mRNA levels in the septum and hippocampus; (2) a down-regulation of TrkA and p75NTR and an up-regulation of TrkB; (3) reduced susceptibility to develop chemical kindling; (4) recovery of the PTZ-induced changes in the expression of neurotrophin receptors in the septal and hippocampal tissues. This data clearly indicates that CCK-induced variation of neurotrophin synthesis in brain is able to influence the susceptibility to develop seizures in adult rats most probably by counteracting the progressive neuronal dysfunction and/or damage.

507 Effect of chemical kindling model induced by PTZ on CCK MRNA and CCK-LI levels in rat brain regions

507 Effect of chemical kindling model induced by PTZ on CCK MRNA and CCK-LI levels in rat brain regions

Effect of chemical kindling model induced by PTZ on CCK mRNA and CCK-LI levels in rat brain regions

An increased expression of cholecystokinin (CCK) messenger RNA (mRNA) as well as CCK-B receptor mRNA in dorsal root ganglion (DRG) cells following peripheral axotomy has previously been demonstrated. In the present in vivo microdialysis study, the effect of unilateral sciatic nerve section on basal and potassium-induced release of CCK-like (CCK-LI) immunoreactivity in the rat dorsal horn was investigated. We also compared the effects of the CCK-B receptor antagonist CI988 on basal and potassium-stimulated CCK-LI release in intact animals and in chronically axotomized rats. Perfusion of the microdialysis probe with KCl (100 mM) induced a more than 6-fold increase of the extracellular level of CCK-LI in control animals. In contrast, following unilateral sciatic nerve section the same KCl stimulation failed to evoke a release of CCK-LI ipsilaterally. However, after systemic administration of CI988 (1 mg kg−1, i.v.), 100 mM KCl induced a significant increase of the extracellular CCK-LI level in axotomized rats, similar to that observed in control animals. In control animals no effect of CI988 on KCl-stimulated CCK-LI release could be detected. CI988 by itself had no influence on the extracellular CCK-LI level in either nerve injured or control animals. The present data suggest that axotomy reduces the release of CCK-like immunoreactivity in the spinal cord by a mechanism involving the CCK-B receptor binding site.